Cyclosporine-insensitive mode of cell death after prolonged myocardial ischemia: Evidence for sarcolemmal permeabilization as the pivotal step

<div><p>A prominent theory of cell death in myocardial ischemia/reperfusion (I/R) posits that the primary and pivotal step of irreversible cell injury is the opening of the mitochondrial permeability transition (MPT) pore. However, the predominantly positive evidence of protection against infarct afforded by the MPT inhibitor, Cyclosporine A (CsA), in experimental studies is in stark contrast with the overall lack of benefit found in clinical trials of CsA. One reason for the discrepancy might be the fact that relatively short experimental ischemic episodes (<1 hour) do not represent clinically-realistic durations, usually exceeding one hour. Here we tested the hypothesis that MPT is not the primary event of cell death after prolonged (60–80 min) episodes of global ischemia. We used confocal microcopy in Langendorff-perfused rabbit hearts treated with the electromechanical uncoupler, 2,3-Butanedione monoxime (BDM, 20 mM) to allow tracking of MPT and sarcolemmal permeabilization (SP) in individual ventricular myocytes. The time of the steepest drop in fluorescence of mitochondrial membrane potential (ΔΨ_m)-sensitive dye, TMRM, was used as the time of MPT (T_MPT). The time of 20% uptake of the normally cell-impermeable dye, YO-PRO1, was used as the time of SP (T_SP). We found that during reperfusion MPT and SP were tightly coupled, with MPT trending slightly ahead of SP (T_SP-T_MPT = 0.76±1.31 min; p = 0.07). These coupled MPT/SP events occurred in discrete myocytes without crossing cell boundaries. CsA (0.2 μM) did not reduce the infarct size, but separated SP and MPT events, such that detectable SP was significantly ahead of MPT (T_SP -T_MPT = -1.75±1.28 min, p = 0.006). Mild permeabilization of cells with digitonin (2.5–20 μM) caused coupled MPT/SP events which occurred in discrete myocytes similar to those observed in Control and CsA groups. In contrast, deliberate induction of MPT by titration with H₂O₂ (200–800 μM), caused propagating waves of MPT which crossed cell boundaries and were uncoupled from SP. Taken together, these findings suggest that after prolonged episodes of ischemia, SP is the primary step in myocyte death, of which MPT is an immediate and unavoidable consequence.</p></div

Assessment of infarct size using TTC staining.

<p><b>A</b>, <i>left to right</i>, examples of original scanned images of TTC staining, the respective green channel signal, and the thresholded regions, respectively (see detailed description of TTC staining procedure in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s002" target="_blank">S1 Fig</a>). Rows from top to bottom in <b>A</b> represent <i>No_BDM</i>, <i>Control</i>, and <i>CsA</i> groups, respectively. <b>B</b>, average percent of severe infarct (white), intermediate infarct (pink) and viable tissue (red) in the three experimental groups. The error bars are omitted for clarity, but none of the regions were significantly different between the groups by 2-way ANOVA. At least in part this is due to the large variation in TTC staining data between hearts in each group (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s007" target="_blank">S6 Fig</a>).</p

Wave-like propagation of MPT caused by H₂O₂.

<p>The time is shown according to the onset of observation starting 6 min after the end of H₂O₂ application (200 μM). <b>A</b> to <b>D</b>, consecutive snapshots taken every 2 minutes. Green, fluorescence of TMRM (F_TMRM); orange, fluorescence of YO-PRO1 (F_YO-PRO1). The nature of objects brightly stained with YO-PRO1 is unknown, but they may represent dead fibroblasts or other cell types which are usually abundant in the most superficial layers and are visible immediately after YO-PRO1 delivery to the heart. White arrows point to two such bright objects, which were used as fiducial points to track myocytes amid slight changes in the field of view and the focal plane. Note that unlike the naturally occurring MPT/SP events, H₂O₂-induced MPT propagated as a wide front (white line), easily crossing cell boundaries, and was not associated with immediate cellular YO-PRO1 uptake. <b>E</b> and <b>F</b>, Different structure of the propagating front of _FTMRM loss during naturally occurring MPT (<b>E</b>; same cell as #13 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s005" target="_blank">S4 Fig</a> as well as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.s014" target="_blank">S5 Movie</a>) and H₂O₂-induced MPT (<b>F</b>). In each Panel, F_TMRM was computed as the function of the distance along the long axis of the rectangular region of interest (ROI) approximating the selected cell (white rectangle). The magnified image of the ROI is shown at the top of each Panel. For each x-position along the cell, F_TMRM was averaged over all pixels in the respective column (across the width) of the rectangle. This yielded an instantaneous profile of the propagating wave (green curve). The minimal value on the y-axis of mean F_TMRM represents the minimal value in the field of view (extracellular level, or background). Note that during naturally occurring MPT (<b>E</b>) the level of F_TMRM in the wake of the MPT front is very close to the background level indicating complete loss of the TMRM in that part of the cell. In contrast, during H₂O₂-induced MPT (<b>F</b>) the level of F_TMRM in the wake of the MPT front remains at about 50% of the dynamic range.</p

The summary of the time difference between the MPT and SP events (T_SP-T_MPT) in individual myocytes from the three experimental groups as indicated.

<p>Thick horizontal bars show average values. Note that CsA increases separation between MPT and SP, such that T_SP-T_MPT is significantly negative. *, p < 0.01.</p

Detailed spatiotemporal analysis of F_TMRM loss and F_YO-PRO1 gain during reperfusion in a representative myocyte from a control heart.

<p><b>A</b>, each row shows F_TMRM (green), F_YO-PRO1 (orange) and the merged image of the cell undergoing the critical transition (outlined with white) for different time points (<i>a</i> to <i>e</i>) indicated in <b>B</b>. <b>B</b>, the cell-averaged F_TMRM (green) and F_YO-PRO1 (orange) as the function of time shown as 0–100% of the dynamic range of each signal. The vertical green and orange dashed lines indicate T_MPT and T_SP, respectively. Note the spatiotemporal overlap between the processes of F_TMRM loss (indicator of MPT) and F_YO-PRO1 gain (indicator of SP), such that at time point <i>b</i> there is a clear uptake of YO-PRO1 while the majority of the cell interior still shows polarized mitochondria.</p

Detailed spatiotemporal analysis of F_TMRM loss and F_YO-PRO1 gain during reperfusion in a myocyte from a heart treated with CsA (0.2 μM).

<p>Notations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.g001" target="_blank">Fig 1</a>. Note that compared to a myocyte from a control heart shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200301#pone.0200301.g001" target="_blank">Fig 1</a>, the earliest detectable F_YO-PRO1 gain (indicator of SP), which is observed at the time point <i>b</i>, clearly occurs before the processes of F_TMRM loss (indicator of MPT).</p

Metabolic Determinants of Electrical Failure in Ex-Vivo Canine Model of Cardiac Arrest: Evidence for the Protective Role of Inorganic Pyrophosphate

<div><p>Rationale</p><p>Deterioration of ventricular fibrillation (VF) into asystole or severe bradycardia (electrical failure) heralds a fatal outcome of cardiac arrest. The role of metabolism in the timing of electrical failure remains unknown.</p> <p>Objective</p><p>To determine metabolic factors of early electrical failure in an Ex-vivo canine model of cardiac arrest (VF+global ischemia).</p> <p>Methods and Results</p><p>Metabolomic screening was performed in left ventricular biopsies collected before and after 0.3, 2, 5, 10 and 20 min of VF and global ischemia. Electrical activity was monitored via plunge needle electrodes and pseudo-ECG. Four out of nine hearts exhibited electrical failure at 10.1±0.9 min (<i>early-asys</i>), while 5/9 hearts maintained VF for at least 19.7 min (<i>late-asys</i>). As compared to <i>late-asys</i>, <i>early-asys</i> hearts had more ADP, less phosphocreatine, and higher levels of lactate at some time points during VF/ischemia (all comparisons p<0.05). Pre-ischemic samples from <i>late-asys</i> hearts contained ∼25 times more inorganic pyrophosphate (PPi) than <i>early-asys</i> hearts. A mechanistic role of PPi in cardioprotection was then tested by monitoring mitochondrial membrane potential (ΔΨ) during 20 min of simulated-demand ischemia using potentiometric probe TMRM in rabbit adult ventricular myocytes incubated with PPi versus control group. Untreated myocytes experienced significant loss of ΔΨ while in the PPi-treated myocytes ΔΨ was relatively maintained throughout 20 min of simulated-demand ischemia as compared to control (p<0.05).</p> <p>Conclusions</p><p>High tissue level of PPi may prevent ΔΨm loss and electrical failure at the early phase of ischemic stress. The link between the two protective effects may involve decreased rates of mitochondrial ATP hydrolysis and lactate accumulation.</p> </div

Heat map (<i>Left</i>) and fold expression (<i>Right</i>) of metabolomic screening of pre-LDVF hearts.

<p>For % filled value, the number of samples, in which each metabolite was detected, was divided by the total number of baseline samples (n = 15). For fold expression, x-axis indicates the ratio of the levels of metabolites in late-asys over those from early-asys hearts. (*:p<0.05).</p

Effect of PPi on ΔΨm during near-anoxia in cultured rat neonatal cells.

<p><b>A–C</b>. Control. ΔΨm-dependent signal gradually decreased throughout 10 min of anoxia. <b>D–F.</b> PPi-treated cell. During the first 5 min of anoxia the PPi-treated cell showed the transient increase of the TMRM fluorescence, indicating the hyperpolarization of ΔΨm, which was followed by attenuated ΔΨm depolarization between 5 and 10 min. <b>G.</b> Quantitative analysis of the TMRM signals.</p

Effect of PPi on ΔΨm during anoxia+tachypacing in rabbit adult ventricular myocytes.

<p>The myocytes were paced at CL = 280 ms for 5 min in a normal HEPES-buffered perfusion solution. After that myocytes were exposed to 20 min of anoxia with continuous pacing at CL = 280 ms (anoxia+tachypacing). <b>A</b> and <b>B</b>. The image of TMRM fluorescence immediately before and after 20 min of anoxia+tachypacing in a representative control myocyte, respectively. Note that after 20 min of anoxia+tachypacing the brightness of ΔΨm-dependent TMRM signal markedly decreased. <b>C</b> and <b>D.</b> The image of TMRM fluorescence immediately before and after 20 min of anoxia+tachypacing in a representative PPi-treated myocyte, respectively. Note that the brightness of ΔΨm-dependent TMRM signal was largely preserved during 20 min of anoxia+tachypacing in this myocyte. <b>E.</b> Statistical analysis of the TMRM fluorescence during 5 minutes of tachypacing followed by 20 minutes of tachypacing+anoxia in control (n = 9) and PPi-treated (n = 7) myocytes. The measurements were normalized to the baseline value recorded at immediately before the initiation of tachypacing (at the time point “−5 min”). ΔΨm-dependent TMRM signal was significantly different between control and PPi groups at all time points between 2 and 20 of tachypacing+anoxia (p<0.05).</p